SARS-CoV-2 humoral and cellular immunity following different combinations of vaccination and breakthrough infection

The elicited anti-SARS-CoV-2 immunity is becoming increasingly complex with individuals receiving a different number of vaccine doses paired with or without recovery from breakthrough infections with different variants. Here we analyze the immunity of individuals that initially received two doses of mRNA vaccine and either received a booster vaccination, recovered from a breakthrough infection, or both. Our data suggest that two vaccine doses and delta breakthrough infection or three vaccine doses and optionally omicron or delta infection provide better B cell immunity than the initial two doses of mRNA vaccine with or without alpha breakthrough infection. A particularly potent B cell response against the currently circulating omicron variant (B. 1.1.529) was observed for thrice vaccinated individuals with omicron breakthrough infection; a 46-fold increase in plasma neutralization compared to two vaccine doses (p < 0.0001). The T cell response after two vaccine doses is not significantly influenced by additional antigen exposures. Of note, individuals with hybrid immunity show better correlated adaptive immune responses compared to those only vaccinated. Taken together, our data provide a detailed insight into SARS-CoV-2 immunity following different antigen exposure scenarios.

Line 183: revise title, "Not only the number but also the type of immunization is important for potent humoral immunity against the SARS-CoV-2" The study only describes mRNA immunization, in this case again the authors term immunization with recovery from infection.

Line 185: And Ref, suggest Khoury Nat Med 2020.
Line 224: The authors say the N antibody data complies with/reflects the S1 specific data but where does it say what % volunteers seroconverted to S1-specific IgG? Fig1b shows significant differences but not number/% volunteers. This information could be inferred by counting the dots but it would be good to have this information specifically shown and/or the N antibody data presented in the same way as Fig1b. Also the authors state that the baseline is the 2xvacc but it would be good to see the data presented against the pre-pandemic levels (WHO standard).
Line 253. Delete 'partially true', suggest: "In contrast to plasma, the only significant correlations in saliva were …" Fig2d: Y axis % of IgG -> should be Ig? How can IgG be less than 100% of itself? Are the colours flipped or is this proportion of Ig? Again need legends for these figures: e.g. Fig2e what does the shading indicate? Line 263: The FACS gating strategy and gate frequency should be added as supplementary, especially for the definition of memory B cells (Fig 3a, 4a). Line 269/270: I don't agree with this conclusion is correct for IgM ie 2xvaccdelta looks very similar to 2xvacc even with variability. I think best to state no statistical differences seen, though there was a trend with some vaccine combinations such as IgA 2xvaccdelta, 3vacc and put in discussion that this might be due to large intraindividual variability. Line 277/278: Statistics needed for support. Line 287: delete "the (change to "protection from severe disease"). Figure 4: Further detail needed in legends-median and IQR shown? Line 297: need to be careful with wording given no significance. Say a trend not that they were higher. Line 300: Again can't say it was highest as not statistically significant. Also need to tell us what the medians were. Line 307-308: Is it meant that infection groups don't develop N-specific T cell responses? Line 312-318: Stats needed to support. Line 319: Put in ref to fig4h after "functions". Line 326-327: Again these conclusions need stats to back them up (from above paragraph).

Figure 5 annotation: Did you use both Pearson's and Spearman's as the figure legend mentions both and the heatmap label is not titled with either.
Discussion: Line 359: Given the timing of infection has not been disclosed, it is problematic to refer to B cell memory and T cell memory responses based on a sampling timepoint as memory rather than phenotype against Omicron/Delta etc. Unless the assertion is that these are crossreactive vaccine induced memory B cells based on the timepoint of sampling (which is not specificed). Or if the cells have been phenotyped for memory -please include gating strategy. Please clarify. Line 362: "responses" Line 364: change to "of SARS-CoV-2" Ref for this statement (line 364/365). Line 370 add in "neutralized the omicron virus.."

Line 394: Reference needed
Line 413: This assertion seems to contradict what you said in the results (line 307/308) Line 420: I am not sure you can say that correlated parameters indicate improved immune quality given these volunteers got a breakthrough infection, whereas those in vaccine only groups without correlations presumably didn't get any breakthrough infections over the same time period may actually have superior immunity as they were protected? You are implying that they will then have a subsequent improved immune quality based on other literature but you need to acknowledge that your findings could equally point to the opposite.
This discussion needs to include strengths and limitations discussion of the study, rather than repeating a lot of the findings. One e.g. of a limitation is alpha neutralisation was not assessed. Also it was an observational study not randomised etc.
Reporting summary: Population characteristics: Please justify that participants were not discriminated based on demographics as this information not included in the paper Recruitment: States "random employees".. does this mean volunteers were randomised? I don't think so as this was an observational study Replication: Please clarify in the manuscript where technical replicates were performed and where not and justify/explain when they were not used Data collection: Please include these dates in the manuscript Flow cytometry: Please include all missing data on flow cytometry plots in figures (axis labels, axis scales, gate frequencies).

Reviewer #3 (Remarks to the Author):
This is an interesting and well-written manuscript defining the antibody and cellular immune responses to COVID-19 mRNA vaccination alone or in combination with breakthrough infections. Despite the low sample size in some groups (n=7), the authors find differences between immunization groups, showing more robust responses after omicron and delta breakthroughs. More specifically, the authors describe better antibody and B cell responses with 2 vaccine doses and delta breakthrough infection or three vaccine doses and omicron or delta infection than only 2 doses of vaccine with or without alpha breakthrough infection. Regarding T cell immunity, no differences were detected by the diverse immunization groups and overall T cell responses were low. A strength of the manuscript is that the variants of the breakthrough infections were identified by sequencing.

Main concerns:
-I disagree with the conclusion of the paper (line 427) that is also stated in the introduction (line70) regarding the 4th immunization not boosting the immune response except for the omicron breakthrough infection. The results do not support this statement as it is written and it may be misleading. In order to show a lack of boosting, the post-immunization response should be compared with the response just before the last immunization.
-Time between immunizations has an impact on the acquired immune response. This fact seems to be ignored in the manuscript. The alpha infections occurred very early after primary vaccination with the two doses. This is totally different from the groups having delta or omicron breakthroughs many months after primary infection. This should be acknowledged in the discussion as could explain why alpha breakthrough seems to induce poorer responses.
-There is no demographic and clinical information on the study participants and comparison between the study groups of the main demographic variables that have been associated with COVID-19 and immune responses. There may be confounders.
-Individuals were sampled 2-9 weeks following the last immunization. Were there differences between the immunization groups in the time since immunization? This could be a confounder too. -The authors conclude that breakthrough infections induce better coordination of the immune response because responses are better correlated. However, is observing stronger correlations in plasma determinations really a reflection of better coordination of the acquired response? I would be more cautious with the statements related to bettercoordinated responses.

-In the introduction, the authors refer many times to the protection of COVID-19 vaccines against infection, while current licensed COVID-19 vaccines are designed to protect against disease. It is well known that protection against infection and decrease of transmission by vaccination is poor. Booster doses are not needed to curb the SARS-CoV-2 transmission as it is mentioned in line 48, but to protect against severe disease. Also, the sentence in lines 57-58 does not make much sense because any infection would boost responses and the fact of having a breakthrough infection already means that responses were not optimal at that time.
Minor comments: -Line 370: a couple of commas would increase clarity

REVIEWER COMMENTS 1 2
Reviewer #1 (Remarks to the Author): In the manuscript "SARS-CoV-2 immunity following different combinations and breakthrough 5 infections" Pusnik et al. investigate the adaptive immune response after different combinations of 6 vaccinations (two vs. three doses) in the presence or absence of breakthrough infection. In addition, 7 they also investigate the immune response after breakthrough infection with different variants of 8 concerns (alpha, delta, and omicron). A scenario very much reflected in current real life. Importantly, 9 the variant of concern was determined by RT-PCR rather than inferring it from the time point of 10 infection. Overall, this is a very important and timely study as this answers the question if the times 11 of antigen exposure and/or the type of exposure (vaccination vs. natural infection) influences 12 adaptive immune responses. 13 14 Minor comments:

15
Reviewer: All samples were collected 2-9 weeks following the last immunization. Please clarify that 16 this time point also refers to breakthrough infections. i.e., 2 weeks after infection…. It would be 17 important to show that the average time point within these 2-9 weeks was comparable between 18 groups, as the adaptive immune response can change significantly within this time point. 19 Response: The reviewer raises an important point. With the term "immunization", we wanted to 20 describe any type of challenge triggering an immune response including infection. However, we 21 agree that this might be confusing since immunization is often understood as a synonym for 22 vaccination. We now exchanged the term "immunization" for "antigen contact" and point out that it 23 covers both infection and vaccination throughout the article. 24 The individuals belonging to different immunization groups were preselected so that the times from 25 the last immune challenge, let it be infection or vaccination, did not significantly differ between the 26 groups (see graph below). We now added this information to the manuscript. 27 28 29 30 Reviewer: It would be interesting to compare the 3+Vacc+O group to a group that has been 31 vaccinated four times, as this would most accurately reflect the 4-time antigen exposure in both 32 groups. 33 Response: We agree with the reviewer, however, we were not able to collect samples of 4-times 34 vaccinated individuals during the study, since the 4 th dose was not recommended for the general 35 population in Germany but rather for individuals at risk (e.g. elderly, pre-existing conditions).  2). Although the flow cytometry panels were 46 different, one can appreciate that only infected individuals had notably more IFNg-expressing N-47 specific CD4 T cells than the individuals from the current study that were initially vaccinated and 48 afterward acquired an infection. This is an important observation and was added to the discussion. 49 We thank the reviewer for his comments. 50 51 52 Reviewer #2 (Remarks to the Author): The study by Pusnik et al presents a comprehensive immunogenicity combined vaccine with 55 breakthrough infection study. However, there are concerns below about cohort detail that 56 undermine the strength of the findings, as my main issue is that the timing of infection plays a bigger 57 role than the antigenic difference but is not sufficiently described-is the timepoint of sampling after 58 infection is comparable between groups (2-9 weeks?).The methods are sufficiently described. Some 59 statistical correlations are not adequately described. FACS gating strategies and gate frequencies are 60 not given. 61 62 Main comment:

63
Reviewer: Cohort details are needed for subject details as booster doses are recommended for at 64 risk groups which may differ from vaccine only groups. 65 Response: We agree with the reviewer and provide the cohort details as a supplemental table 1. The 66 third vaccination was recommended for the general population in Germany at the time of study and 67 most of the vaccinated individuals also received the third dose of vaccine regardless of their risk 68 status. Therefore, to our knowledge, there is no bias towards the high-risk population in any of the 69 groups. This information has now been added to the discussion. Moreover, none of the study 70 participants indicated to be immunodeficient. Two individuals in the 2xVacc group and one in the 71 3xVacc group were noted to have taken cortisol which acts immunosuppressive, however, their 72 immune responses were normal. 73 74 Reviewer: The timing between vaccination and infection is also likely different between the subjects 75 infected with different variants and therefore their boosting potential is either related to timing post 76 vaccination (i.e., infection at a short-term memory versus long term memory when neutralising 77 antibodies have waned is likely to recruit different immune parameters) and/or antigenic distance 78 and immune evasion from the ancestral vaccine. Furthermore, the sample timepoint after infection is 79 not given (e.g. N positivity rates after alpha infection-is this due to long sample timepoint?) and 2-9 80 weeks after infection samples were compared? 81 Response: The reviewer raises an important point. The timing between different immune challenges 82 let it be vaccination or infection is indeed different between the different groups since it reflects the 83 real-world situation where the onset of vaccination campaigns and prevalence of different SARS-CoV-84 2 variants occurred at different times (see figure 1a). It was for example not possible to get samples 85 from vaccinated individuals that would acquire alpha and omicron infection at the same time after 86 vaccination since the omicron variant appeared much later than the alpha and most individuals got 87 vaccinated at the beginning of spring 2021. Therefore, we do not claim in the manuscript that we 88 compared the immunogenicity of different variants causing breakthrough infections, but rather 89 compare the immune response of individuals that underwent different immunization scenarios at a 90 given time after the last immunization. Together with antigenic distance, the severity of infection and 91 immunogenicity the time between immunizations contribute to the differences in immune response 92 observed for the groups assessed in the study. We added this important point to the discussion and 93 made it clear that we do not claim a comparison of immunogenicity against different variants. 94 The term immunization was used to describe any type of immune challenge including vaccination 95 and infection (We now exchanged the term "immunization" for "antigen contact" and point out that 96 it covers both infection and vaccination throughout the article), so 2-9 weeks apply to both 97 vaccination and infection as the last immunization. The individuals belonging to different 98 immunization groups were preselected so that the times from the last immune challenge, let it be 99 infection or vaccination, did not significantly differ between the groups (see graph below). We now 100 added this information to the manuscript. 101 102 103 Reviewer: Often immunisation is used to refer to vaccine or recovery from infection as a form of 104 immunization. This is confusing for the reader as immunisation typically means vaccine. If you want 105 to use the term this way throughout the article (including the way you refer to your different groups) 106 then this needs to be flagged in the introduction for clarity to the reader. The difference between 107 hybrid immunity (infection then vaccination) or breakthrough infection (vaccination then infection) 108 and immunization does not equate to vaccination or recovery from infection but vaccination 109 however is used interchangeably in the text. 110 Response: We thank the reviewer for pointing out this issue. The term immunization was used in the 111 manuscript to describe any immune challenge; either vaccination or infection. We now exchanged 112 the term "immunization" for "antigen contact" and point out that it covers both infection and 113 vaccination throughout the article. 114  190 191 Reviewer: Line 64 Change to "amongst individuals" Change "the" to "their" or delete. Response: We removed this section from the introduction and will only discuss it in the discussion. 215 The higher magnitude of immune responses at the time of sampling was meant by "more robust 216 immunity". 217 Please look at the response to major comment #2. 218 219 Reviewer: Methods: 220 Line 75: "Individuals"? -cohort details are needed for age/gender/timepoints/controls and date of 221 recruitment/study period. 222 What were the third and fourth vaccinations? Were these also mRNA vaccines? What determined if 223 they had third and fourth vaccines or didn't? age groups or underlying conditions have been 224 prioritised for boosters-the cohort should be specified. sampling were added as supplemental figure 1 due to the lack of space in Figure 1. Figure 1a is a 258 schematic representation of the immunization and sampling for different groups. It shows the 259 sequential order and approximate length of periods between the immunization and sampling events. 260 Detailed information is now provided as supplemental material. 261 Please also look at the response to major comment #2. 262 263 Reviewer: Fig1c and 1g for Omicron figures should have the same axis as the other two (scale 10-264 10,000 vs 100-10,000) 265 Response: We believe tha this does not make sense since the neutralization of different variants is 266 compared in the next figure (1d). Y-axis ticks are automatically adjusted to visualize the data in the 267 best resolution (changing the axis to 100-10000 would compress the data points and would be 268 harder to distinguish; particularly bad for groups 2xVacc and 2xVacc+a). Reviewer: Line 183: revise title, "Not only the number but also the type of immunization is important 274 for potent humoral immunity against the SARS-CoV-2" 275 The study only describes mRNA immunization, in this case again the authors term immunization with 276 recovery from infection. 277

Response:
The term immunization was used in the manuscript to describe any immune challenge; 278 either vaccination or infection. We now exchanged the term "immunization" for "antigen contact" 279 and point out that it covers both infection and vaccination throughout the article. Response: We thank the reviewer for the suggestion, the reference was added. 283 284 Reviewer: Line 224: The authors say the N antibody data complies with/reflects the S1 specific data 285 but where does it say what % volunteers seroconverted to S1-specific IgG? Fig1b shows significant 286 differences but not number/% volunteers. This information could be inferred by counting the dots 287 but it would be good to have this information specifically shown and/or the N antibody data 288 presented in the same way as Fig1b. 289 Also the authors state that the baseline is the 2xvacc but it would be good to see the data presented 290 against the pre-pandemic levels (WHO standard). 291 Response: Since the participants were all initially 2-times vaccinated they all had detectable anti-292 Spike antibodies. This is now evident from supplemental table 1. For the anti-Nucleocapsid 293 antibodies, we only have information on whether the individuals were positive or negative since the 294 assay used is not quantitative. 295 The pre-pandemic levels are considered negative results since all used antibody determination assays 296 use measurements of plasma from individuals collected before the pandemic as a cutoff defining 297 seropositivity. 298 299 Reviewer: Line 253. Delete 'partially true', suggest: "In contrast to plasma, the only significant 300 correlations in saliva were …" 301 Response: We changed the sentence accordingly 302 Response: There should be Ig instead of IgG, we corrected the mistake. The shading around the line 307 represents the 95% confidence interval around the line of best fit, we now improved the figure  308 legends. 309 310 Reviewer: Line 263: The FACS gating strategy and gate frequency should be added as supplementary, 311 especially for the definition of memory B cells (Fig 3a, 4a). 312 Response: The gating strategies have been added as supplemental figures 2 and 3. 313 314 Reviewer: Line 269/270: I don't agree with this conclusion is correct for IgM ie 2xvaccdelta looks very 315 similar to 2xvacc even with variability. I think best to state no statistical differences seen, though 316 there was a trend with some vaccine combinations such as IgA 2xvaccdelta, 3vacc and put in 317 discussion that this might be due to large intraindividual variability. 318 Response: We agree with the reviewer, and changed the sentence accordingly. 319